The present invention provides methods and compositions for the alteration of compounds produced by secondary metabolic pathways in plants. The invention also provides plant cells modified in content of secondary metabolites and plant seed with altered secondary metabolite content. In one embodiment, the content of anti-nutritional secondary metabolic products is altered in plants, plant cells and plant seeds according to the invention. In another embodiment, products found within the phenylpropanoid and sugar alcohol secondary metabolic pathways are altered in plants, plant cells and plant seeds according to the invention. The invention further provides genetic constructs and vectors useful for modifying the secondary metabolite content of plant cells and seeds. The invention further relates to modified seed meal, and to animal feed containing modified seed meal, particularly seed meal in which the secondary metabolite content is reduced or altered.
Plants produce a variety of compounds by way of secondary metabolism. While not considered essential to plant metabolism, secondary metabolic pathways often produce unique biochemicals, some of which are considered anti-nutritional or even toxic. The secondary metabolic pathways and the compounds produced by these pathways are be specific to an individual species or genus. Thus manipulation of secondary metabolic pathways can produce novel compositions of biochemicals or produce plant tissue with altered secondary metabolic content. In particular, the manipulation of secondary metabolism for the purpose of alteration of secondary metabolic compounds that are anti-nutritional or toxic in nature can provide unique applications in the food and feed area.
It is desirable to manipulate secondary metabolism without disturbing the biochemical processes considered essential for plant cell growth and survival. The collection of biochemical processes and the compounds involved which are essential for the growth and survival of the plant, are considered primary metabolic pathways and their products. Primary metabolism is generally considered to encompass those biochemical processes that lead to the formation of primary sugars, (such as glucose), amino acids, common fatty acids, nucleotides and the polymers derived from them (polysaccharides such as starch, proteins, lipids, RNA and DNA etc.) Yeoman and Yeoman, Tansley Review No. 90, Manipulating Secondary Metabolism in Cultured Plant Cells, New Phytologist, 134:553-569, 1996.
Thus the art recognizes that primary metabolism can be defined as those metabolic processes essential to the survival and growth of all plant cells whereas secondary metabolism can be defined as those biochemical processes that are not essential to all plant cells. For example, secondary metabolic. pathways determine such plant features as colour, taste, morphology, etc. Secondarymetabolism also produces various compounds that are recognized by insects or are involved in pathogen response. Some of these compounds may provide a benefit to some plant species under wild conditions, but under cultivation these compounds may be detrimental to the quality of the harvested product or may restrict the utility of the crop for certain applications. Some of the secondary metabolites are unique compounds that have evolved within a species as a result of specialized biochemical pathways. Secondary metabolism is not characterized by the redundancy in biochemical mechanisms which is typical of primary metabolism, thus, characteristically, the products of secondary metabolism are not produced by multiple pathways in the plant. Secondary metabolites are typically more plant-specific than the ubiquitous biochemicals which are involved in the primary pathways.
Numerous attempts to manipulate primary metabolic pathways have resulted in plant cells with altered starch or oil (lipid) content. However, gross manipulation of primary metabolism can be expected to lead to deleterious effects. For example, the composition of lipids can be changed, but elimination of lipids would obviously be deleterious to cell survival. Manipulation of primary metabolism is not always completely successful because redundant biochemical mechanisms can overcome some attempts at manipulation. Thus primary metabolic pathways in plants are often difficult to manipulate in a fashion that is predictable and provides useful and tangible results under cultivation conditions.
In some instances, primary metabolism has been altered successfully to produce a novel phenotype which represents a compositional change rather than a reduction or elimination of a specific substance. Typically, these manipulations have been accomplished by ectopic expression of a plant gene, such as over-expressing a gene in certain tissues or in a constitutive fashion rather than a regulated fashion, or by inhibition of a specific gene activity by antisense RNA, ribozymes or co-suppression. However, it has been difficult to predict a priori the results of such manipulations.
The expression of a plant enzyme can be modified at many levels. This includes control at the gene expression level, translation, protein processing and allosteric control of protein function. Thus ectopic expression of a plant gene involved in primary metabolism may not overcome the complex biochemical controls on regulation of primary metabolism. Furthermore, redundancy in primary metabolism also poses a difficult hurdle to overcome in these manipulations since primary metabolic pathways are essential to plant growth and survival. Accordingly attempts to alter primary metabolism often fail to provide the intended phenotype. Moreover, the evaluation of these modified plants at the field level, or under a variety of environmental extremes has often led to the discovery that the predictedeffect is not observed or plant performance is compromised. Thus, modification of primary metabolism requires careful consideration ofthe primary metabolic pathway or the discreet step in a pathway in order to achieve a specific phenotype.
The manipulation of secondary metabolic pathways has been complicated by a poor understanding of the biochemistry involved, little information on the genes expressed in secondary metabolic pathways and the complex inter-relationships between biochemical pathways in general.
However, methods to alter secondary metabolism can provide a valuable means to produce novel phenotypes, including those with altered levels of secondary metabolic compounds, for example those considered anti-nutritional in nature. Thus secondary metabolic pathways represent an important target for the genetic manipulation of plants.
Two biochemical pathways in plants that are considered secondary metabolic pathwayshave been the subject of studies aimed at alterationof the levels of the final products. The methods used to manipulate these pathways have not produced the desired results. For example, the phenylpropanoid pathway is involved in the formation of lignin and is considered a secondary metabolic pathway. The biosynthesis of lignin is part of the general phenylpropanoid biosynthetic pathway which produces at least three primary phenolic precursors, coumaric, ferulic and sinapic acids, products of which are polymerized into lignin and other phenolic compounds (see FIG. 2).
In attempts to alter the secondary metabolic phenylpropanoid pathway, the genes for many of the enzymes involved in the formation of the lignin monomers are currently identified as targets for lignin reduction via antisense or co-suppression technologies (e.g. U.S. Pat. Nos. 5,451,514, 5,633,439, WO 93/05160, WO 94/08036). These target genes include those encoding cinnamyl alcohol dehydrogenase, caffeic acid O-methyl-transferase and phenylalanine ammonia lyase. These techniques are directed to reduction of lignin content as this is assumed to have an overall beneficial effect on processing or digestibility of plants.
However, reduction of lignin by antisense or co-suppression technologies by targeting one of the genes in the phenylpropanoid pathway may have a number of undesirable effects. These may include increased disease susceptibility, altered growth rates or reduction of the physical strength of the plant fibre and hence a reduction in agronomic performance. It was shown that inhibition of the enzyme phenylalanine ammonia lyase leads to numerous deleterious phenotypes (Elkind et al., Abnormal Plant Development and Down-Regulation of Phenylpropanoid Biosynthesis in Transgenic Tobacco Containing a Heterologous Phenylalanine Ammonia Lyase Gene, Proc. Natl. Acad. Sci. USA 87: 9057-9061, 1990). The enzyme phenylalanine ammonia lyase acts on the primary metabolite, phenylalanine, an amino acid. The results from these experiments demonstrate that the alteration of secondary metabolism through modification of one of the primary metabolites involved in a particular secondary metabolic pathway may produce unexpected and deleterious phenotypes. Accordingly, the choice of the biochemical step(s) within a secondary metabolic pathway is crucial to producing plants which are phenotypically normal but which show a reduction of a specific secondary metabolite. Furthermore, the use of antisense RNA or co-suppression strategies may not provide the level or specificity of secondary metabolite reduction that is commercially acceptable. Additionally, inhibition of the genes encoding key enzyme activities may affect the expression of related genes. Thus little progress has been made with reduction of phenolic compounds considered anti-nutritional, or reduction of lignin content by antisense RNA or co-suppression without accompanying deleterious side effects.
A second example of efforts to alter a metabolic pathway that failed to produce the desired results is modification of glucosinolate biosynthesis in canola. Attempts to modify glucosinolate content of canola meal by manipulation of the glucosinolate pathway have been reported. One method that has been proposed to alter the biosynthesis of glucosinolates was to create a new pathway that competes for the sulfur used in the formation of glucosinolates, or reduce levels of tryptophan used in the formation of glucosinolates by conversion of tryptophan to tryptamine (xe2x80x9cEngineering Altered Glucosinolate Biosynthesis by Two Alternative Strategiesxe2x80x9d, by Ibrahim, Chavadej and De Luca, published in: Genetic engineering of plant secondary metabolism 1994, Plenum Publishing Corporation; New York; USA).
However, the method was not successful in reducing glucosinolate content of canola seed meal. The method failed to reduce the anti-nutritional content of glucosinolates in canola meal. Glucosinolates are made in the leaves of the plant and then transported to the seed. Thus the method was based on the belief that the simple alteration of the availability of one of the primary metabolites (sulphur, the amino acid tryptophan) used in the formation of glucosinolates would reduce the production of glucosinolates. However, the primary glucosinolates in seed are aliphatic glucosinolates that do not utilize the amino acid tryptophan for the production of side chains. Moreover, the results from these experiments (e.g., Chavadej et al., Proc. Natl. Acad. Sci USA, 91: 2166-2170, 1994) demonstrated that transgenic plants that carried an enzyme capable of altering the primary amino acid tryptophan did not contain reduced glucosinolates in the seed, the aliphatic glucosinolate content in the seed was equal to or possibly even greater than non-tranagenic plants. Thus the production of total glucosinolates in seed was not reduced even though a minor component (indole glucosinolates) appeared to be reduced. It is clear from genetic studies that low glucosinolate plants can be obtained by conventional breeding and there are a number of loci controlling low glucosinolates in crucifers. There are numerous biochemical transformations that take place within glucosinolate biosynthesis and those steps in common with all or most of the synthesis of glucosinolates are the steps which need to be targetted if a method of reducing total glucosinolates in needed. Thus, a general method to alter glucosinolate production in crucifers must take into account the different enzymes and substrates used in glucosinolate biosynthesis if a method of general utility is to be devised.
However, it appears that the enzymes used to obtain this modification also acted upon primary metabolites (the animo acid tryptophan and the mineral sulfur) thus any significant alteration of these compounds in the plant cell would be expected to have a deleterious effect. Accordingly the proposed method failed to specifically target the secondary metabolic pathway. Indeed the alteration of tryptophan can be expected to lead to many deleterious effects. Thus, alteration of the levels of a primary metabolite did not produce the intended effect of low glucosinolate canola meal, underscoring the difficulty of modifying primary metabolism.
Accordingly, a general method to alter secondary metabolism will be valuable for altering the biochemical composition of plant tissues. This can include, for example, reduction of anti-nutritional compounds, alteration of secondary metabolic profiles, providing plant tissue with altered processing characteristics, alteration of the levels of compounds of industrial utility or pharmaceutical interest, production of plants with modified taste, texture or appearance, production of plants with altered secondary metabolites involved in insect attraction, disease tolerance or other biological processes that are influenced by secondary metabolites, or plants with growth characteristics positively modified by alteration of secondary metabolites.
The present invention provides a method to target the formation of a secondary metabolite. The method comprises altering the availability of a substrate that is specific to the secondary metabolic pathway and essential to the formation of the final secondary metabolic product, particularly those compounds within one to five biochemical steps of final product formation. Targeting substrates at steps near the final product formation avoids the problems associated with alterations of metabolites also involved in primary pathways, since it occurs after the entry point of a substrate into the primary metabolic pathway. Thus the method provides a novel means of specifically targeting the reduction or alteration of secondary metabolites by identifying precursors used within a secondary metabolic pathway that do not comprise substrates for primary metabolic pathways.
The method can also comprise altering the availability of a substrate in a tissue specific manner such that only certain tissues, e.g. seed tissues, are altered.
Thus the method provides a novel means of specifically targeting the reduction or alteration of secondary metabolites by identifying precursors used within a secondary metabolic pathway that do not comprise primary metabolic compounds.
In one embodiment, the present invention provides a method of making a genetically transformed plant comprising:
A) introducing into a plant cell capable of being transformed and regenerated to a whole plant a DNA expression cassette comprising, in addition to DNA sequences required for transformation and selection in plant cells, a DNA sequence that, under the control of a promoter active in plant cells, encodes a protein capable of modifying the utilization of a substrate in a secondary metabolic pathway, with the proviso that the substrate is not a primary metabolite of the group selected from glucose, amino acids, common fatty acids and nucleotides, and
B) recovering a plant which has an altered content of at least one product of the secondary metabolic pathway.
In another embodiment, the present invention provides a method for making a genetically transformed seed comprising growing the plant obtained according to steps A and B of the method described above under conditions which permit the formation of seed.
The recombinant DNA is chromosomally integrated into the genome of a fertile plant mush that it is passed to subsequent generations.
In further embodiments, the present invention provides vectors for transforming plants, plants and seeds transformed according to the method described above, and feed products containing the seeds or meal derived therefrom.